The long-term objective of this program is the determination of the sequence of molecular events during the duplication of Escherichia coli, and the biochemical mechanisms which control the initiation and completion of each of the major events in the sequence. The major objectives of the current project are the determination of the molecular mechanisms which control the initiation of DNA replication. Four projects will be undertaken in the next year. 1) We suggest that the timing of initiatton of chromosome replication in E. coli B/r may be determined by the length of an envelope-attached segment of the chromosome at the origin and the rate of membrane elongation along this segment. As a partial test of this model, the relationship between cell volume and initiation of chromosome replication will be analyzed using an osmotic-sensitive mutant of Salmonella typhimurium and spheroplasts of B. megaterium to determine the effects of cellular swelling on the induction of initiation of chromosome replication in the presence and absence of inhibitors of RNA and protein synthesis. 2) As a further test of this model, we will attempt to isolate mutants of E. coli B/r possessing longer than normal initiation times in a given growth medium employing the bacteriophage Mu to obtain mutation through integration of the phage. If such mutants can be obtained, the location of the incorporated phage and its relationship to the origin of chromosome replication will be determined. 3) The timing of replication of an F'lac plasmid in three stocks of E. coli B/r (A, F and K) growing at numerous rates will be determined in order to evaluate how the timing of synthesis of this plasmid varies with respect to chromosome replication as the growth rate of the cells is varied. 4) We plan to initiate a program to measure the relationship between synthesis of specific cellular proteins and initiation of chromosome replication, including total membrane proteins, membrane proteins at the site of origin attachment to the cell membrane and the pattern of synthesis of the dnaC gene product during the division cycle.